Human ABO Blood Group-Binding Lactobacilli

ABSTRACT

Lactobacillus  screening methods were carried out using surface plasmon resonance spectrums and human intestinal mucin and blood group antigens as probes. A trial to set selection criteria in the above-mentioned methods of screening for lactobacilli was made to adapt the methods to mass screening, and it was discovered that lactobacilli compatible with ABO blood groups can be screened by setting 100 RU as a criterion for judging bacterial binding under certain conditions. Using 238  lactobacillus  strains, the above-mentioned screening methods and tests to judge their compatibility for the use of yogurt production were carried out, to at long last specifically discover  bacillus  strains compatible with blood groups A, B, and O.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/720,462, filed Aug. 24, 2007; which is a National Stage Applicationof International Application Number PCT/JP2005/022096, filed Dec. 1,2005; which claims priority to JP 2004-349135, filed Dec. 1, 2004 andalso claims priority to International Application NumberPCT/JP2005/011043, filed Jun. 16, 2005; all of which are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to lactobacilli and screening oflactobacilli.

BACKGROUND ART

About 200 species, 100 trillion (10¹⁴) or more intestinal bacteria(intestine-colonizing microorganisms) inhabit the human intestine.Microorganisms called probiotics improve the intestinal balance betweenuseful and harmful bacteria, contributing to host health. Recently, atrend is to apply these probiotic microorganisms to foods. For example,several kinds of functional yogurt produced using lactobacilli withprobiotic functions have been commercialized. Thus, a mass screeningtechnique for selecting more excellent probiotics is required.

Intestine-colonizing lactobacilli propagate while adhering to the humanintestine. Thus, the property of lactobacilli to bind to the intestineis very important for the exertion of probiotic functions. The bindingmechanism of lactobacilli in the human intestine is not yet elucidated.Prior studies on intestinal lactobacilli have confirmed thatLactobacillus casei has the ability to bind to sugar chains ofglycolipids and that L. reuteri and L. crispatus have a collagen-bindingability. In addition, lectin-like proteins which bind to theabove-mentioned intestinal lactobacilli have been identified. However,cytoskeleton protein (collagen)-exposed areas are very few in theintestinal epithelia of most healthy individuals, and colonization oflactobacilli via lectin-like proteins in the intestinal epithelia isunlikely. Thus, sugar chains that bind to intestinal mucin areconsidered to play an important role in ability of intestine-colonizinglactobacilli to bind to the intestine. Surface layer proteins (SLPs) ofmany intestine-colonizing lactobacilli have lectin-like proteins, whichare sugar-recognizing proteins. Intestinal mucin exists on intestinalsurface.

Intestinal mucin is a mucous high-molecular-weight glycoprotein havingcountless mucin-type sugar chains linked to a polypeptide (a coreprotein, apomucin) via O-glycosidic linkages. In sum,intestine-colonizing lactobacilli are considered to acquireintestine-binding ability by binding to sugar chains of intestinal mucinthrough lectin-like proteins on their surface and establishing a stablegrowth.

Meanwhile, an interesting fact that has recently been reported is thatthe chemical structure of sugar chains constituting human colonic mucin(HCM) varies depending on the ABO blood group (Non-Patent Documents 1 to4).

Human ABO blood groups are distinguished depending on the type ofantigenic substance expressed on red blood cell surface. The antigenicsites of these ABO blood group substances are sugar chains of certainchemical structures (ABO blood group antigens). Both blood group A- andB-antigens are molecules consisting of three sugars. The blood groupA-antigen is a molecule in which an α-N-acetylgalactosamine is bound toa basic structure called blood group H antigen that consists of twosugars through a specific linkage mode, whereas the blood group Bantigen is a molecule in which α-galactose is bound. Humans of bloodgroup A, blood group B, and blood group AB express A antigen, B antigen,and both A and B antigens on the surface of red blood cells,respectively. In contrast, humans of blood group O express H antigen,which is the basic structure.

The above scientific fact that the sugar chain structure ofdigestive-tract mucin varies blood group-dependently suggests that thetype of probiotic lactobacilli that bind to and grow in the digestivetract varies depending on the blood group. Development of functionalyogurt tailored at individual levels will be made possible iflactobacilli that are compatible with each blood group are found.Focusing on this point, the present inventors have hitherto developed amethod of screening for human intestine-binding lactobacilli using theiradsorbability to ABO blood group antigens (Patent Document 1). This isan epoch-making method that detects adsorbability of lactobacilli to ABOblood group antigens by using surface plasmon resonance (SPR) spectrums,and thereby selecting compatible lactobacilli according to blood groups.Specifically, by using ABO blood group antigens or intestine-derivedmucin as ligands, the method detects the binding between lactobacilliand the ligands occurring when the lactobacilli are contacted with theligands immobilized on a sensor chip, through detecting a mass change onthe sensor chip which accompanies the binding as a surface plasmonresonance (SPR) signal. The above-mentioned mass change is expressed byresonance units (RU). One RU equals 1 pg/mm², and means that 1 pg of asubstance is bound per 1 mm². The present inventors carried out theabove method, and confirmed that Lactobacillus crispatus JCM8778 strainand Lactobacillus acidophilus OLL2769 strain recognize blood group Aantigen (Patent Document 1 and Non-Patent Document 5). However, anincreased demand for foods that use probiotic lactobacilli includingyogurt is expected, and thus acquisition of lactobacilli having bloodgroup specific binding capabilities and with better binding propertieshas been awaited.

-   [Patent Document 1] Japanese Laid Open Patent Application No.    2004-101249 (unexamined, published Japanese patent application)-   [Non-Patent Document 1] Junko Amano, Seikagaku, The Japanese    Biochemical Society, 1999, Vol. 71, p. 274-277-   [Non-Patent Document 2] Holgersson, J., Stromberg, N., and    Breimer, M. E., Glycolipids of human large intestine: glycolopid    expression related to anatomical localization,    epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes    of the donors. Biochimie, 70, 1565-1574 (1988).-   [Non-Patent Document 3] Holgersson, J., Jovall, P. A., and    Breimer, M. E., Glycosphingolipids of human large intestine:    detailed structural characterization with special reference to blood    group compounds and bacterial receptor structures. J. Biochem,    (Tokyo), 110, 120-131 (1991).-   [Non-Patent Document 4] Vanak, J., Ehrmann, J., Drimalova, D.,    Nemec, M., monoclonal antibodies in the detection of blood group    antigens A and B in the mucosa of the large intestine. Cas Lek Cesk,    18, 364-367 (1988).-   [Non-Patent Document 5] Uchida, H. et al., Biosci. Biotechnol.    Biochem., 68(5), 1004-1010 (2004).-   [Non-Patent Document 6] Holmes, S. D. et al., Studies on the    interaction of Staphylococcus aureus and Staphylococcus epidermidis    with fibronectin using surface plasmon resonance (BIACORE)., J.    Microbiological Methods, 28, 77-84 (1997).-   [Non-Patent Document 7] Fratamico, P. M. et al., Detection of    Escherichia coli O157:H7 using a surface plasmon resonance    biosensor. Biotechnol. Techniques. 7, 571-576 (1998).

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances. Aproblem to be solved by the present invention is to discover novelintestine-binding lactobacilli compatible with human ABO-blood groups.

Means for Solving the Problems

To solve the above-mentioned problems, the present inventors carried outthe above-mentioned lactobacilli screening method that utilizes surfaceplasmon resonance spectrums. The above-mentioned method has already beenestablished as a screening method for lactobacilli, but the presentinventors set “selection criteria values” in the above-mentionedscreening method for lactobacilli in order to make the method moreadaptable to mass screening. Examples of prior studies on bacteria usingsurface plasmon resonance spectrums, are where binding betweenStaphylococcus aureus and fibronectin was measured by using surfaceplasmon resonance spectrums (Non-Patent Document 6) and whereEscherichia coli O0157 was detected by using protein A or protein Gimmobilized on a chip via anti-Escherichia coli 0157 antibodies(Non-Patent Document 7). The RU values indicating binding of livebacteria were from about 100 to 1,000 RU in these studies. However, noexample of an investigation of lactobacilli using surface plasmonresonance spectrums was known except for the reports by the presentinventors (Patent Document 1 and Non-Patent Document 5). Furthermore, RUvalues could vary depending on various measurement conditions even iftest samples were the same. Thus, the present inventors conducteddedicated research, and as a result, established screening methods forABO blood group-compatible lactobacilli, by setting 100 RU as acriterion under certain conditions.

Moreover, the present inventors carried out the above-mentionedscreening methods on 238 separate human intestine-derived lactobacillusstrains isolated from human intestine, further conducted tests to judgetheir suitability for use in yogurt production, and at long lastspecifically discovered bacillus strains compatible with blood groups A,B and O. Namely, the present invention relates to lactobacilli suitablefor blood group-compatible yogurt and screening methods for thelactobacilli, and specifically provides the following inventions:

[1] An intestine-binding lactobacillus Lactobacillus gasseri, having theability to bind to a human ABO blood group antigen represented by anyone of the following formulas of:

(a) [GalNAcα1-3(Fucα1-2)Gal-];

(b) [Galα1-3(Fucα1-2)Gal-]; and

(c) [Fucα1-2Gal-].

[2] The lactobacillus Lactobacillus gasseri of [1], wherein thelactobacillus is specified by any one of Accession No.: NITE BP-25,Accession No.: NITE BP-26, Accession No.: NITE BP-27, Accession No.:NITE BP-28, Accession No.: NITE BP-145, and Accession No.: NITE BP-146.[3] A starter for producing a human ABO blood group-compatible fermentedmilk and dairy product, comprising the lactobacillus Lactobacillusgasseri of [1] or [2].[4] A food and drink product, comprising the lactobacillus Lactobacillusgasseri of [1] or [2].[5] A fermented milk and lactic acid bacteria beverage, comprising thelactobacillus Lactobacillus gasseri of [1] or [2].[6] A method of screening for a lactobacillus using a surface plasmonresonance spectrum, wherein fulfillment of the following conditions (i)to (iii) is used as an index:

(i) RU value indicating ability to bind to a human ABO blood group Aantigen is 100 RU or higher;

(ii) RU value indicating ability to bind to a human ABO blood group Band/or H antigen is 100 RU or lower; and

(iii) RU value indicating ability to bind to human blood group Aintestinal mucin is 100 RU or higher.

[7] A method of screening for a lactobacillus by using a surface plasmonresonance spectrum, wherein fulfillment of the following conditions isused as an index:

(i) RU value indicating ability to bind to a human ABO blood group Bantigen is 100 RU or higher;

(ii) RU value indicating ability to bind to a human ABO blood group Aand/or H antigen is 100 RU or lower; and

(iii) RU value indicating ability to bind to human blood group Bintestinal mucin is 100 RU or higher.

[8] A method of screening for a lactobacillus by using a surface plasmonresonance spectrum, wherein fulfillment of the following conditions isused as an index:

(i) RU value indicating ability to bind to a human ABO blood group Hantigen is 100 RU or higher;

(ii) RU value indicating ability to bind to a human ABO blood group Aand/or B antigen is 100 RU or lower; and

(iii) RU value indicating ability to bind to human blood group Ointestinal mucin is 100 RU or higher.

[9] A method of screening for a lactobacillus by using a surface plasmonresonance spectrum, wherein fulfillment of the following conditions isused as an index:

(i) RU value indicating ability to bind to a human ABO blood group Bantigen is 100 RU or higher;

(ii) RU value indicating ability to bind to a human ABO blood group Aand/or H antigen is 100 RU or lower; and

(iii) RU value indicating ability to bind to human blood group Bintestinal mucin is higher than an RU value indicating ability to bindto human blood group A intestinal mucin and an RU value indicatingability to bind to human blood group O intestinal mucin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the chemical structure of biotinylatedpolymer (BP) probes that have sugar chain antigen regions of human ABOblood groups.

FIG. 2 is a drawing showing the sugar chain portion of a blood group Aantigen BP probe (top), the sugar chain portion of a blood group Bantigen BP probe (middle), and the sugar chain portion of a blood groupO antigen BP probe (bottom).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides intestine-binding lactobacilliLactobacillus gasseri having the ability to bind to human ABO bloodgroup antigens that are represented by any one of the formulas of (a)[GalNAcα1-3(Fucα1-2)Gal-], (b) [Galα1-3(Fucα1-2)Gal-], and (c)[Fucα1-2Gal-].

“Lactobacilli” is generally a collective designation for a group ofbacteria that produce lactic acid from glucose with a yield of 50% ormore, when expressed by molar ratio conversion. Genus Lactobacillus,genus Lactococcus, genus Streptococcus, and genus Leuconostoc arerepresentative genera of lactobacillus. Genus Bifidobacterium is alsoincluded in the lactobacillus of the present invention. GenusLactobacillus is further classified into species. Representativebacteria species of genus Lactobacillus comprise Lactobacillusdelbruekii subsp. bulgaricus (L. bulgaricus), Lactobacillus delbruekiisubsp. delbruekii (L. delbruekii), lactobacilli of the group ofLactobacillus acidophilus (L. acidophilus group), Lactobacillus casei(L. casei), Lactobacillus plantarum (L. plantarum), Lactobacillus brevis(L. brevis), Lactobacillus buchneri (L. buchneri), Lactobacillusfermentum (L. fermentum), Lactobacillus helveticus (L. helveticus), andsuch. Depending on the results of DNA-DNA homology and cell wallcomponent analysis, lactobacilli of L. acidophilus group are classifiedinto six species: Lactobacillus acidophilus (A-1), Lactobacilluscrispatus (A-2), Lactobacillus amylovorus (A-3), Lactobacillusgallinarum (A-4), Lactobacillus gasseri (B-1), and Lactobacillusjohnsonii (B-2).

The lactobacilli Lactobacillus gasseri (herein below sometimesabbreviated as “L. gasseri”) of the present invention areintestine-binding Lactobacillus gasseri characterized by the ability tobind to ABO blood group antigens. In the present invention, ABO bloodgroup antigens refer to sugar chains that determine blood groups, andspecifically, refer to blood group A sugar chain (A antigen):[GalNAcα1-3 (Fucα1-2)Gal-], blood group B sugar chain (B antigen):[Galα1-3(Fucα1-2)Gal-], and blood group O sugar chain (refers to thesugar chain determining group O blood; also called H antigen or Oantigen): [Fucα1-2Gal-]. As mentioned above, intestinal mucin present onintestinal surface have different sugar chains depending on the ABOblood group. Among lactobacilli that bind to mucin prepared from bloodgroup A human intestine, the present inventors confirmed the existenceof lactobacilli L. gasseri that bind to blood group A sugar chains, theantigen determining human blood group A. Further, the present inventorsconfirmed that blood group A sugar chains are expressed in theabove-described mucin. Thus, the lactobacilli L. gasseri of the presentinvention that bind to the above-mentioned blood group A sugar chain,but which are not adsorbed onto sugar chains of other blood groups, bindto the blood group A sugar chains present on intestinal mucin, therebyacquiring intestine-binding ability in humans of the ABO blood group A.These lactobacilli L. gasseri are considered to stably bind to andproliferate in the intestine of blood group A individuals, leading tothe exertion of probiotic functions. Namely, these lactobacilli L.gasseri can contribute to the health of, especially, blood group Aindividuals, and can be said to be compatible with blood group Aindividuals. Similarly, the lactobacilli L. gasseri of the presentinvention that bind to the above-mentioned blood group B sugar chain,but which are not adsorbed onto sugar chains of other blood groups, arecompatible with ABO blood group B individuals. The lactobacilli L.gasseri of the present invention that bind to the above mentioned bloodgroup O sugar chain, but which are not adsorbed onto sugar chains ofother blood groups are compatible with ABO blood group O individuals.

The lactobacilli L. gasseri of the present invention (occasionallycalled below as “blood group-specifically-binding lactobacilli L.gasseri”) can be isolated/separated from human feces. It is highlypossible that lactobacilli L. gasseri compatible with blood group Aindividuals (abbreviated below as “group A-compatible lactobacilli”) canbe more efficiently isolated from feces of ABO blood group Aindividuals, lactobacilli L. gasseri compatible with blood group Bindividuals (abbreviated below as “group B-compatible lactobacilli”)from feces of ABO blood group B individuals, and lactobacilli L. gassericompatible with blood group O individuals (abbreviated below as “groupO-compatible lactobacilli”) from feces of ABO blood group O individuals.For isolation, properties of lactobacilli L. gasseri known to thoseskilled in the art can be used as indicators. For example, beingbacillary, homofermenting, exhibiting aerobic growth, having no gasproduction, and such are usable as indicators.

Media generally suitable for culturing lactobacilli may be used forculturing the blood group-specifically-binding lactobacilli L. gasseriof the present invention, and media comprising carbon sources such asglucose, lactose, galactose, fructose, trehalose, sucrose, mannose, andcellobiose; nitrogen sources such as meat extracts, peptone, yeastextracts, casein, and whey proteins; and inorganic nutrients such asmagnesium sulfate, ferrous sulfate, and manganese sulfate can be used.As one preferable example, Lactobacilli MRS broth (Difco, Ref No.288130) can be given. The culture conditions are not especiallyrestricted, as long as the growth of enteric lactobacilli is permitted.Preferable conditions include, for example, pH 5.0-pH 8.0 andtemperature of 20° C.-45° C., and more preferable conditions areanaerobic, pH 5.0-pH7.0, and temperature of 30° C.-40° C.

One can know whether or not the L. gasseri isolated and cultured asmentioned above have blood group-specific binding ability to theintestine or binding ability to blood group antigens, by determining thepresence or absence of their ability to bind to human intestinal mucinor blood group antigens. For example, bacterial surface layer proteins(SLPs) can be prepared from the surface of test bacteria, and thebinding between SLPs labeled with biotin and such and intestinal mucinor blood group antigens can be detected. Alternatively, detection by thehybridization technique using labeled intestinal mucin or labeled bloodgroup antigens after electrophoresis of the test bacteria's SLPs is alsopossible. Further, as in the later-mentioned Examples, the detection canbe carried out with lactobacilli in a living state if a surface plasmonanalytical apparatus (for example, BIACORE1000) is utilized.

To prepare human blood group-specific intestinal mucin, surface portionsof human intestine of a certain blood group can be obtained, theportions subjected to gel filtration using a solubilizing agent such asguanidine hydrochloride, and then purified by using both a highabsorbance of proteins and high content of neutral sugars as indicators.For instance, one can conduct such processes by referring to the methoddescribed in “Purushothaman, S. S. et al., Adherence of Shigelladysenteriae 1 to Human Colonic Mucin. Curr. Microbiol., 42(6), 381-387(2001).” It is more preferable if the expression of blood group antigensin the prepared human intestinal mucin is confirmed by using anti-bloodgroup antigen antibodies. The method described in Examples is given as aspecific example. Meanwhile, with regard to blood group antigens, theymay be synthesized based on the later-described formulas or sugarsequences described in FIG. 2, or use commercially available sugar chainprobes (example: probes made by Seikagaku Corporation) orneoglycoprotein/blood group A-trisaccharide BSA (example: Calbiochem),neoglycoprotein/blood group B-trisaccharide BSA (example: Calbiochem),and such.

Representative blood group-specifically-binding lactobacilli L. gasseriof the present invention comprise Lactobacillus gasseri specified by anyone of Accession No.: NITE BP-25, Accession No.: NITE BP-26, AccessionNo.: NITE BP-27, and Accession No.: NITE BP-28. Among them, thelactobacilli specified by Accession No.: NITE BP-26 and Accession No.:NITE BP-27 are group A-compatible lactobacilli, and those specified byAccession No.: NITE BP-25 and Accession No.: NITE BP-28 are groupO-compatible lactobacilli. These bacterial strains are lactobacilli thatwere confirmed by the present inventors to be compatible with theabove-mentioned each blood group. Further representativeblood-group-specifically-binding lactobacilli L. gasseri of the presentinvention comprise Lactobacillus gasseri that are specified by AccessionNo.: NITE BP-145 or Accession No.: NITE BP-146. These lactobacillispecified by Accession No.: NITE BP-145 and Accession No.: NITE BP-146are group B-compatible lactobacilli. These bacterial strains wereconfirmed by the present inventors to be compatible with blood group B.

The present inventors deposited these bacterial strains with the PatentMicroorganisms Depositary (NPMD) of the National Institute of Technologyand Evaluation according to the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. Herein below, the contents specifying the deposit aredescribed.

The contents for Accession Nos. NITE BP-25, 26, 27, and 28 are asfollows.

(A) The Institute of deposition: NPMD (Location: 2-5-8, Kazusakamatari,Kisarazu-city, Chiba, 292-0818, Japan)(B) Original depositary date: Oct. 8, 2004

Date of receipt of request for conversion of the original deposits todeposits under the Budapest Treaty (depositary conversion date): May 6,2005

(C) Accession Nos.:

Lactobacillus gasseri OLL2915 strain (Accession No.: NITE BP-25)

Lactobacillus gasseri OLL2804 strain (Accession No.: NITE BP-26)

Lactobacillus gasseri OLL2818 strain (Accession No.: NITE BP-27)

Lactobacillus gasseri OLL2827 strain (Accession No.: NITE BP-28)

The contents for Accession Nos.: NITE BP-145 and 146 are as follows.

(A) The Institute of deposition: NPMD (Location: 2-5-8, Kazusakamatari,Kisarazu-city, Chiba, 292-0818, Japan)(B) Original depositary date: Oct. 4, 2005

Date of receipt of request for conversion of the original deposits todeposits under the Budapest Treaty (depositary conversion date): Nov.25, 2005

(C) Accession Nos.:

Lactobacillus gasseri OLL2877 strain (Accession No.: NITE BP-145)

Lactobacillus gasseri OLL2901 strain (Accession No.: NITE AP-146)

Further, the Lactobacillus gasseri deposits will be stored and madeavailable to the public in accord with the provisions of the BudapestTreaty for the Deposit of Microorganisms, i.e., they will be stored withall the care necessary to keep them viable and uncontaminated for aperiod of at least five years after the most recent request for thefurnishing of a sample of the deposit, and in any case, for a period ofat least thirty (30) years after the date of deposit or for theenforceable life of any patent which may issue disclosing the culture.The depositor acknowledges the duty to replace the deposits should thedepository be unable to furnish a sample when requested, due to thecondition of the deposits. During pendency of this application, accessto the deposits will be afforded to one determined by the Commissionerto be entitled thereto. All restrictions on the availability to thepublic of the subject culture deposits will be irrevocably removed uponthe granting of a patent disclosing them.

The blood group-specifically-binding lactobacilli L. gasseri of thepresent invention are usable for producing food and drink productscompatible with each blood group. The food and drink products producedby using the blood group-specifically-binding lactobacilli L. gasseri ofthe present invention are not restricted in terms of their category andkinds, and may be functional foods, foods for specified health use,health foods, and nursing care foods, or may be confectioneries, lacticacid beverages, dairy products such as cheese and yogurt, seasonings,and such. Forms of the food and drink products are also not restricted,and can be any form of foods and drinks that are usually distributablesuch as solid-, liquid-, liquid-formula-like-, jelly-like-, tablet-,granule-, or capsule-form. The above-mentioned food and drink productscan be produced by usual methods known to those skilled in the art. Inthe above-mentioned production of food and drink products, one can addsugars, proteins, lipids, dietary fibers, vitamins, trace metalsessential for the living body (manganese sulfate, zinc sulfate,magnesium chloride, potassium carbonate, and such), flavorings, andother additives, as long as the growth of lactobacilli is not prevented.

The blood group-specifically-binding lactobacilli L. gasseri of thepresent invention are not only usable when mixed with general foods anddrinks but are also usable as starters for the production of,especially, dairy products and fermented milk such as yogurt and cheese.When used as starters, other microorganisms may be mixed in as long asthe inhabitation and proliferation of the bloodgroup-specifically-binding lactobacilli L. gasseri of the presentinvention and the production of dairy foods are not adversely affected.For example, Lactobacillus delbruekii subsp. bulgaricus, Streptococcusthermophilus, Lactobacillus acidophilus, and such, which are the mainbacterial species of lactobacilli for yogurt, can be mixed in and alsoother bacterial species that are generally used for yogurt and cheesecan be mixed in to give starters. Yogurt production using theabove-mentioned starters can be carried out by usual methods. Forinstance, plain yogurt can be produced by mixing the above-mentionedstarters with milk or dairy products that have been cooled after beingheated, mixed, homogenized, and sterilized, and then fermenting andcooling the mixture.

The food and drink products produced by using the bloodgroup-specifically-binding lactobacilli L. gasseri of the presentinvention comprise the lactobacilli. When a human individual of acompatible ABO blood group consumes the food and drink products, it isexpected that the blood group-specifically-binding lactobacilli L.gasseri of the present invention contained in the food and drinkproducts will bind to the intestine and proliferate, adjusting andmaintaining the intestinal balance and promptly exerting probioticfunctions. Thus, the food and drink products are useful not only asgeneral foods and drinks but also as functional foods, health andnutrition foods, nursing care foods, health foods, and such foods usedfor health-promoting purposes.

The present invention also provides methods of screening forlactobacilli using surface plasmon resonance spectrums. The screeningmethods of the present invention are for selecting lactobacilli that arecompatible with each blood group. The screening methods of the presentinvention achieve screening by measuring binding ability of lactobacillito blood group-specific human intestinal mucin and ABO blood groupantigens by using surface plasmon resonance spectrums, and carry out theselection using the following points as indicators: (i) having a certainor higher level of binding ability to an ABO blood group antigen of aspecific blood group; (ii) not having a certain or higher level ofadsorption ability to ABO blood group antigens of blood groups otherthan the blood group specified in (i) above; and (iii) having a certainor higher level of binding ability to human intestinal mucin of theblood group specified in (i) above. The measurement of binding abilityto ABO blood group antigens enables the exclusion of those lactobacillithat mainly bind to sites other than antigens determining blood groups(for example, non-terminal partial structures of sugar chains, sitesexposing sialic acids and sulfuric residues, mucin protein portions),and to screen for lactobacilli having a high blood-group specificity. Inthe present invention, human blood group A-intestinal mucin refers tointestinal mucin derived from ABO blood group A individuals. Similarly,human blood group B-intestinal mucin refers to intestinal mucin derivedfrom blood group B individuals and human blood group O intestinal mucinrefers to intestinal mucin derived from blood group O individuals.

In the screening methods of the present invention, an analysis usingsurface plasmon resonance spectrums is carried out by using ABO bloodgroup antigens and human intestinal mucin as probes. As apparatuses foranalyzing interaction among biological molecules using surface plasmonresonance spectrums, one can use, for example, BIACORE1000 (Biacore).The methods for preparing the ABO blood group antigens and humanintestinal mucin are as described above. Known immobilization methodsare usable to immobilize the probes. The method of immobilization may beby physical adsorption or by adsorption via covalent bonds. To give anexample, immobilization can be easily carried out through biotin-avidinbinding, by coating chips with streptavidin and biotinylating theprobes. Commercially available chips that have been streptavidin-coated(Biacore) may also be used.

As surface plasmon resonance spectrums are used in the methods of thepresent invention, the above-mentioned “binding ability” is expressed asresonance units (RU). 1 RU indicates that 1 pg of a substance is boundper 1 mm². In the methods of the present invention, the above-mentioned“certain or higher level of binding ability” is judged using “100 RU” asa criterion. Namely, a judgment that a certain or higher of level/amountlactobacilli are bound to probes is made if a measurement value of “100RU” or more is obtained. For instance, when a measurement value obtainedby using blood group A human-derived intestinal mucin as a probe is 100RU or more, those lactobacilli are lactobacilli that bind to intestinalmucin. RU values could vary depending on measurement conditions.Temperature conditions of the present methods are, for example, 20 to40° C., preferably 20 to 30° C., and more preferably 23 to 28° C.Further, preferable sample concentration of the present methods is 0.1to 0.5 mg/mL, and preferable flow rate of the present methods is 3 to 10μL/min. As long as the conditions are within the above-mentioned ranges,RU values do not vary even if the above-mentioned conditions arechanged; thus one can judge the presence or absence of binding based on“100 RU”. Furthermore, even if various conditions such as sampleconcentrations are changed beyond the above-mentioned ranges, theresulting values are considered to be “100 RU” of the present method aslong as the values are substantially equivalent to 100 RU obtained bythe above-mentioned conditions. With regard to ability to bind to ABOblood group antigen probes, selection may be made by using values otherthan “100 RU” if the purpose is to set a stricter selection criterion.In the methods of the present invention, those bacteria with higher RUvalues are considered to be bacteria that have stronger binding abilityto probes (ligands). Thus, one may screen for bacteria that bind to ABOblood group antigen probes at RU values higher than 100 if the purposeis to obtain bacteria having a stronger binding ability. For example,the value may be 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000RU, and as mentioned later in the Examples, one may screen by using2,000 RU as a criterion. Conversely, with regard to the recognition ofundesired blood groups, one may screen by using values stricter than“100 RU or lower” as a criterion. For example, 90, 80, 70, 60, or 50 RUmay be used as a criterion.

Meanwhile, when the probes in the screening methods of the presentinvention are blood group-specific human intestinal mucin, the bloodgroup-specifically-binding lactobacilli are expected to give measurementvalues (RU) lower than measurement values obtained when ABO blood groupprobes are used. This is because, when human intestinal mucin isimmobilized on a chip, the amount of blood group antigens (sugar chains)bound to the human intestinal mucin is considered to be smaller than theamount obtained when blood group antigens (sugar chains) alone areimmobilized on the chip, in terms of an amount per unit area of thechip. With regard to lactobacilli whose measurement values obtained byusing a probe for a specific ABO blood group are sufficiently high ascompared with values obtained by using a probe for other ABO bloodgroups, they may qualify as the present invention's bloodgroup-specifically-binding lactobacilli even if measurement valuesobtained by using as probes human intestinal mucin of that specificblood group are 100 RU or lower, if they can be judged to have utilityfor the present invention's purposes, by examining whether or notmeasurement values obtained by using as probes the human intestinalmucin of the specific blood group are sufficiently high as compared withthose values obtained by using human intestinal mucin specific for otherblood groups. For example, blood group O-compatible lactobacilli can bescreened by using as indicators the following points: (i) RU valuesindicating a binding ability to human ABO blood group A antigens are 100RU or higher; (ii) RU values indicating binding ability to human ABOblood group B and H antigens are 100 RU or lower; and (iii) RU valuesindicating a binding ability to blood group A human intestinal mucin arehigher than RU values indicating a binding ability to blood group Bhuman intestinal mucin and RU values indicating a binding ability toblood group O human intestinal mucin. Blood group B-compatiblelactobacilli can be screened by using as indicators the followingpoints: (i) RU values indicating a binding ability to human ABO bloodgroup B antigens are 100 RU or higher; (ii) the RU values indicatingbinding ability to human ABO blood group A and H antigens are 100 RU orlower; and (iii) RU values indicating a binding ability to blood group Bhuman intestinal mucin are higher than RU values indicating a bindingability to blood group A human intestinal mucin and RU values indicatinga binding ability to blood group O human intestinal mucin. As for bloodgroup O-compatible lactobacilli, screening is possible by using asindicators the following points: (i) RU values indicating a bindingability to human ABO blood group H antigens are 100 RU or higher; (ii)RU values indicating binding ability to human ABO blood group A and Bantigens are 100 RU or lower; and (iii) RU values indicating a bindingability to blood group O human intestinal mucin are higher than RUvalues indicating a binding ability to blood group A human intestinalmucin and RU values indicating a binding ability to blood group B humanintestinal mucin.

In surface plasmon resonance spectrum measurement, RU values maysometime become abnormally high due to bacterial aggregation caused bynon-specific adsorption of test bacteria. As a result of this,adsorption to human-derived intestinal mucin or ABO blood group antigensmay not be correctly reflected on measurement results. Thus, ifabnormally high values are obtained, the lactobacilli under examinationcan be eliminated from screening because non-specific adsorption(bacterial aggregation) is suspected.

Targets of the present invention's screening methods are not especiallyrestricted, as long as they are lactobacilli. If examples of targetswere to be cited, preferably used are Lactobacillus acidophilus,Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillusgallinarum, Lactobacillus gasseri, Lactobacillus johnsonii,Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus zeae,Lactobacillus reuteri, Lactobacillus delbruekii subsp. bulgaricus,Lactobacillus delbruekii subsp. lactis, Lactobacillus fermentum,Lactobacillus murinus, Bifidobacterium animalis, Bifidobacteriumbifidum, Bifidobacterium breve, Bifidobacterium infantis,Bifidobacterium longum, Bifidobacterium pseudolongum, Enterococcusfaecium, Enterococcus fecalis, and Streptococcus thermophilus. Morepreferable target examples are genus Lactobacillus, and the mostpreferable target examples are lactobacilli of Lactobacillus acidophilusgroup, which include L. gasseri.

The lactobacilli screened by the methods of the present invention arethose lactobacilli that are highly likely to bind to human intestine ofa specified ABO blood group. Individuals of the specified blood groupcan promote health by taking in lactobacilli screened by the methods ofthe present invention, thanks to effects such as improvement ofintestinal balance in a short period of time. Therefore, lactobacilliscreened by the methods of the present invention are applicable to foodand drink products, functional foods, foods for specified health use,dairy products, lactic acid bacteria beverages, and such for humanindividuals of a specified blood group.

All prior art references cited in the present specification areincorporated herein by reference.

EXAMPLES

Herein below, the present invention will be specifically explained basedon examples, but it is not to be construed as being limited thereto.

Example 1 Gas Production Test

Lactobacilli that do not produce gas are preferred as lactobacilli usedin yogurt production. This is because Japanese law requires sellingyogurt in airtight containers. This also prevents product defects andruptures due to container expansion accompanying gas production. Thus,gas production tests were carried out with 238 strains.

A lactobacilli activation culture (37° C., 18 hours) was carried outtwice in MRS Broth (DIFCO). Aluminum-capped test tubes each containing aDurham tube and MRS Broth (5 mL) were sterilized (121° C., 15 minutes).Durham tubes were put into the tubes with their openings at the bottomand air bubbles were removed from the Durham tubes during sterilization.10 μL of lactobacillus suspension at about 10⁹ cfu/mL was inoculatedinto each of the above-mentioned sterilized aluminum-capped test tubesand cultured anaerobically at 37° C. for 24 hours. After the end of theculture, presence or absence of gas accumulated in Durham tubes wasvisually observed. When presence of obvious air bubbles was observed,the sample was judged as gas-production positive (+) (Table 1).

Example 2 Analysis of Blood Group Antigen-Recognizing Ability

To obtain lactobacilli for blood group-compatible yogurt, the presentinventors decided to select lactobacilli that recognize and bind toblood group antigens by using surface plasmon resonance spectrums. Astest sample bacteria, 238 strains belonging to the Lactobacillusacidophilus group (L. gasseri, L. plantarum, L. crispatus, L.amylovorus, L. casei, L. salivarius, L. brevis, L. fermentum, and such)were prepared. BIACORE1000 was used as the apparatus for surface plasmonresonance spectrum analysis.

2-(1) Preparation of Analytes

Each bacterial strain was seeded to start culture and passaged threetimes in an MRS medium, and then cultured for 12 hours and dispensedinto 1.5 mL volume tubes in 500 μL aliquots. Bacteria obtained bycentrifugal separation of these suspensions (6,000 rpm at 4° C. for 10minutes) were washed twice in PBS (pH 7.2), freeze-dried, and suspendedat a concentration of 0.1 mg/mL by using HBS-EP buffer (0.01 M HEPES, pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) to give an analytesuspension.

2-(2) Blood Group Antigen Probes and their Immobilization onto Chips

Antigenic structures of human ABO blood group substances were used asprobes for selecting bacteria. Specifically, the following biotinylatedpolymer probes of trisaccharide sugar chains (Seikagaku Corporation)(herein below, also described as “BP-probe(s)”) were used:

A antigen probe: [GalNAcα1-3(Fucα1-2)Gal-]B antigen probe: [Galα1-3(Fucα1-2)Gal-]H antigen probe: [Fucα1-2Gal-]With regard to the above-mentioned BP probes, chemical structures aredepicted in FIG. 1 and sugar chain structures are depicted in FIG. 2.

The above-mentioned blood group antigen BP probes were prepared at 0.1mg/10 mL with HBS-EP buffer (pH 7.4). This solution was added to SensorChip SA (Biacore) and the commercialized probes were immobilized ontothe sensor chip surface via biotin-avidin reaction. Sensor Chip SA is achip with immobilized streptavidin for exclusive use of BIACORE. Theamount of immobilized A antigen probe was set to an amount that gives750 RU, and the amount of immobilized B antigen probe was set to anamount that gives 850 RU. Immobilization of O antigen probes was carriedout as follows: a reaction was carried out in an acetic acid buffer atpH 4.5, at 55° C., for 20 hours by treating 1 mg of A antigen sugarchain probes (Seikagaku Corporation) with 0.5 U of the enzymeα-N-acetylgalactosaminidase (E.C.3.2.1.97, Streptococcuspneumoniae-derived, SIGMA, JAPAN) that hydrolyzes GalNAc existing as anα-linkage, thereby removing the terminal GalNAc of the probes (a removalratio measurement using A antigen antibodies continued that 80% or moreof GalNAc was removed). The biotinylated group H antigen sugar chainprobes were immobilized onto avidin-bound sensor chips in a similarmanner with group A antigen sugar chain probes. The binding amount inthis case was about 1,000 RU.

2-(3) Preparation of Human Intestinal Mucin and their Immobilizationonto Chips

Human colonic mucin was prepared as the other ligand for bacteriaselection. Blood group A human intestine (the colon), blood group Bhuman intestine (the colon), and blood group O human intestine (thecolon) were acquired from the Tohoku University Graduate School ofMedicine as subdivided test sample specimens. Mucus mucin layers werecollected from normal parts of the colons by scraping the surface. Themucin layers were defatted by using Folch's solvent and diethyl etherand dried, followed by an extraction with a 4 M guanidine hydrochloridesolution at 37° C. for two hours. Gel filtration was carried out and theobtained filtrates were used for tests as purified human blood group A,B, or O intestinal mucin (human colonic mucin: HCM, herein below, bloodgroup A, B, and O intestinal mucin are sometimes abbreviated as A-HCM,B-HCM, and O-HCM, respectively). Purification by gel filtration wascarried out according to the human colonic mucin purification methoddescribed in “Purushothaman, S. S. et al, Adherence of Shigelladysenteriae 1 to Human Colonic Mucin. Curr. Microbiol., 42(6), 381-387(2001)”. 4M guanidine hydrochloride solution was used as mobile phaseand a Toyopearl HW-65F column (90 cm×2.6 cm, Tosoh, Tokyo, Japan) wasused. With regard to the detection, neutral sugars and proteins weremeasured by a phenol-sulfuric acid method (490 nm) and by absorbance at280 nm, respectively. Fractions with protein absorbance and highestneutral sugar content peaks were selected. These were furtherfractionated based on a molecular weight of about 2,000,000 or more andwere isolated as human colonic mucin (HCM) preparations. The obtainedHCMs were confirmed for human blood group substrate antigenicity of eachblood group by using antibodies, The sampling of the above-mentionedspecimens was carried out with the approval of the Ethical Committee ofthe Tohoku University Graduate School of Medicine, and the consent ofpatients.

Immobilization of HCMs to chips for BIACORE1000 was carried out by anamine coupling method. First, a mixed reagent solution consisting of 50μL of 75.0 mg/mL N-ethyl-N′-(3-dimethyl aminopropyl-carbodiimidehydrochloride) (EDC) and 50 μL of 11.5 mg/mL N-hydroxysuccinimide (NHS)was run over the sensor chip CM5 to which a carboxymethyl dextran grouphas been introduced in advance, and thereby the carboxyl groupsintroduced at the dextran termini were activated. A mixed solution of120 μL of acetic acid buffer (pH 4.0) for immobilization and 30 μl ofHCM-A was run over them, and the HCM was covalently bound theretothrough an amine coupling reaction. Next, remaining active groups atsites where no ligands were bound were blocked by using a 1Methanolamine hydrochloride-NaOH solution (pH 8.5). HBS-EP buffer wasused as running buffer. The amount of immobilized HCM was set to 1,000to 2,000 RU so that the screening methods of the present invention canbe conducted in a highly reproducible manner.

2-(4) Measurement by BIACORE1000

An interaction of the above-mentioned analytes with blood group antigenswas analyzed with BIACORE1000 by using the above-mentioned chips withimmobilized blood group antigen probes or chips with immobilized HCM.The BIACORE1000 measurement conditions are shown below:

Running buffer solution: HBS-EP buffer (pH 7.4)

Loaded sample volume: 20 μL

Flow rate: 3 μL/min

Temperature: 25° C.

Regeneration solution: 5 μL of 1 M guanidine hydrochloride solution

In BIACORE analyses, the interaction between analytes and blood groupantigens is expressed by resonance units (RU). One resonance unitindicates the binding of 1 pg substance per 1 mm². Results are shown inTable 1.

Selection of blood group A-compatible lactobacilli A antigen- A antigen-recognizing recognizing Stomach- A antigen- B antigen- ability/ Hantigen- ability/ A-HCM- O-HCM- acid recognizing recognizing B antigen-recognizing H antigen- recognizing recognizing Bile-acid toleranceBacterial ability Gas ability recognizing ability recognizing abilityability tolerance (Survival strains (RU) production (RU) ability (RU)ability (RU) (RU) (OD₆₅₀) rate %) MEP 165501 0.0 − 6.5 0.0 956.6 0.0 0.00.0 0.112 0.003 MEP 165502 0.0 − 716.7 0.0 997.2 0.0 0.0 48.0 0.1350.874 MEP 165503 0.0 − 0.0 ∞ 931.1 0.0 0.0 87.0 0.224 1.798 L. gasseri2086.3 − 28.6 72.9 5.1 409.1 829.2 0.0 0.119 24.906 OLL 2804 L. gasseri3981.3 − 52.3 76.1 18.7 212.9 598.2 0.0 0.117 6.212 OLL 2818 Selectionof blood group O-compatable lactobacilli H antigen- H antigen-recognizing recognizing Stomach- H antigen- A antigen- ability/ Bantigen- ability/ O-HCM- A-HCM- acid recognizing recognizing A antigen-recognizing B antigen- recognizing recognizing Bile-acid toleranceBacterial ability Gas ability recognizing ability recognizing abilityability tolerance (Survival strains (RU) production (RU) ability (RU)ability (RU) (RU) (OD₆₅₀) rate %) MEP 165504 14.8 + 7957.2 0.0 3.9 3.838.0 101.4 1.310 0.003 MEP 165505 0.0 − 2415.2 0.0 40.2 0.0 0.0 539.61.164 0.000 MEP 165506 5.3 − 2336.3 0.0 12.5 0.4 0.0 51.3 0.135 0.851 L.gasseri 25761.8 − 32.0 805.1 47.4 543.5 509.0 893.5 0.118 0.959 OLL 2827L. gasseri 1061.7 − 0.0 ∞ 10.9 97.4 6233.0 665.7 0.086 0.585 OLL 2915Selection of blood group B-compatable lactobacilli B antigen- B antigen-recognizing recognizing B antigen- A antigen- ability/ H antigen-ability/ recognizing recognizing A antigen- recognizing H antigen-Bacterial ability Gas ability recognizing ability recognizing strains(RU) production (RU) ability (RU) ability MEP 165507 0 − 224.8 0.0 0 0.0MEP 165508 0 + 0 0.0 96.1 0.0 MEP 165509 0 − 0 0.0 0 0.0 L. gasseri4220.9 − 66.4 63.6 0 ∞ OLL 2877 L. gasseri 2000.9 − 0 ∞ 0 ∞ OLL 2901Selection of blood group B-compatable lactobacilli Stomach- B-HCM-A-HCM- O-HCM- acid recognizing recognizing recognizing Bile-acidtolerance Bacterial ability ability ability tolerance (Survival strains(RU) (RU) (RU) (OD₆₅₀) rate %) MEP 165507 0.0 0.0 2.4 0.339 0.011 MEP165508 18.7 0.0 531.4 2.020 0.010 MEP 165509 0.0 0.0 154.0 0.000 1.499L. gasseri 1406.3 218.2 0.0 0.651 10.812 OLL 2877 L. gasseri 17.1 0.07.6 0.031 8.923 OLL 2901

2-(5) Selection of Bacteria (Part 1)

Lactobacilli were selected according to the results obtained above.First, selection was carried out according to the above-mentionedBIACORE1000 measurement results and the results of gas-production testof Example 1.

Lactobacilli for yogurt for blood group A were selected based on (i)group A antigen-recognizing ability and (ii) gas production. Theselection criteria were:

(i) blood group A antigen-recognizing ability: results obtained byBIACORE1000 measurement using blood group A antigen probes were 2,000 RUor higher (within about top 35%).(ii) gas production: the gas production test showed no gas production(−).By this process, the 238 lactobacillus strains were narrowed down to 43strains.

Similarly, lactobacilli for yogurt for blood group O were selected basedon (i) blood group H antigen-recognizing ability and (ii) gasproduction. The selection criteria were:

(i) blood group H antigen-recognizing ability: results obtained byBIACORE 1000 measurement using group H antigen probes were 700 RU orhigher (within about top 13%).(ii) gas production: the gas production test showed no gas production(−).By this process, the 238 lactobacillus strains were narrowed down to 14strains.

Similarly, lactobacilli for yogurt for blood group B were selected basedon (i) blood group B antigen-recognizing ability and (ii) gasproduction. The selection criteria were:

(i) blood group B antigen-recognizing ability: the results obtained byBIACORE1000 measurement using group B antigen probes were 2,000 RU orhigher (within about top 14%).(ii) gas production: gas production was not seen by the gas productiontest (−).By this process, the 238 lactobacillus strains were narrowed down to 25strains.

2-(6) Selection of Bacteria (Part 2)

As mentioned above, 43 lactobacillus strains were selected aslactobacillus candidates for yogurt for blood group A. These strainsrecognize blood group A antigens and produce no gas. Of these, thecandidates were further selected based on (i) the absence of recognizingability for other blood group antigens (B antigens and H antigens) and(ii) the specific recognition of A antigens. Selection criteria were:

(i) recognizing ability for other blood group antigens (B antigens and Hantigens)B antigen-recognizing ability: results obtained by BIACORE1000measurement using B-antigen probes were 100 RU or lower,H antigen-recognizing ability: results obtained by BIACORE1000measurement using H antigen probes were 100 RU or lower,(ii) Specific recognizing ability for A antigensThe ratio of A antigen-recognizing ability/B antigen-recognizing abilitywas 70 or higher, and the ratio of A antigen-recognizing ability/Hantigen-recognizing ability was 100 or higher.By this process, 13 strains were selected from the 43 strains.

Similarly, lactobacillus candidates for yogurt for blood group O werealso selected. The section criteria were:

(i) recognizing ability for other blood group antigens (A antigens and Bantigens)A antigen-recognizing ability: the results obtained by BIACORE1000measurement using A antigen probes were 100 RU or lower,B antigen-recognizing ability: the results obtained by BIACORE1000measurement using B antigen probes were 100 RU or lower,(ii) Specific recognizing ability for H antigensThe ratio of H antigen-recognizing ability/A antigen-recognizing abilitywas 800 or higher, and the ratio of H antigen-recognizing ability/Bantigen-recognizing ability was 20 or higher. By this process, fivestrains were selected from the 14 strains.

Similarly, lactobacillus candidates for yogurt for blood group B werealso selected. The section criteria were:

(i) recognizing ability for other blood group antigens (A antigens and Hantigens)A antigen-recognizing ability: results obtained by BIACORE1000measurement using A antigen probes were 100 RU or lower,H antigen-recognizing ability: results obtained by BIACORE1000measurement using H antigen probes were 100 RU or lower,(ii) Specific recognizing ability for B antigensThe ratio of B antigen-recognizing ability/A antigen-recognizing abilitywas 50 or higher, and the ratio of B antigen-recognizing ability/Hantigen-recognizing ability was 50 or higher. By this process, 6 strainswere selected from the 25 strains.

2-(7) Selection of Bacteria (Part 3)

The bacterial strains selected above were further selected according tothe recognizing ability for blood group-specific human colonic mucin(HCM).

From the lactobacillus candidates for yogurt for blood group A, (i)those recognizing A-HCM were selected and (ii) those recognizing O-HCMwere eliminated. Specifically, selection criteria were: (i) having 100RU or higher A-HCM-recognizing ability and (ii) having 100 RU or lowerO-HCM-recognizing ability, both from BIACORE1000 measurement resultsusing human HCM. Further, MEP165511 strain and MEP165530 strain showedrecognizing ability at 10,000 RU or higher and were eliminated onsuspicion of bacterial aggregation. According to the above selections,two bacillus strains (L. gasseri OLL 2804 and L. gasseri OLL 2818) wereselected from the 13 strains.

Similarly, from the lactobacillus candidates for yogurt for blood group0, (i) those recognizing O-HCM were selected and (ii) those with arelatively high recognizing ability of A-HCM were eliminated. Namely,selection criteria were: (i) having 100 RU or higher O-HCM-recognizingability and (ii) having 1,000 RU or lower A-HCM-recognizing ability inBIACORE1000 measurement results using human HCM. According to theabove-mentioned selections, two bacillus strains (L. gasseri OLL 2827and L. gasseri OLL 2915) were selected from the five strains.

Similarly, with the lactobacillus candidates for yogurt for blood groupB, the selection criterion was that the B-HCM-recognizing ability ishigher than A-HCM- and H-HCM-recognizing abilities. According to theabove-mentioned selections, two bacillus strains (L. gasseri OLL 2877and L. gasseri OLL 2901) were selected from six strains.

Example 3 Stomach-Acid Tolerance Test and Bile-Acid Tolerance Test

Lactobacilli incorporated into a body in the form of yogurt preferablystay alive in the intestine to fully exert their functions. For thisreason, stomach-acid tolerance test and bile-acid tolerance test werecarried out.

3-(1) Stomach-Acid Tolerance Test

One milliliter bacterial suspension of lactobacilli, prepared by twoactivation cultures (37° C., 18 hours) in Lactobacilli MRS broth (DIFCO)followed by two washes with physiological saline, was added to 9 mL offilter-sterilized artificial stomach acid at pH 2 [NaCl (0.2%), pepsin(1:5000, Tokyo Chemical Industry Co., Ltd) (0.35%): adjusted to pH 2with 1N hydrochloric acid], and kept in contact for two hours underaerobic conditions. After this, 1 mL aliquot was taken from the mixture,and the reaction was stopped by adding 9 mL of 67 mM phosphate buffer(pH 6.5). Numbers of live bacteria before and after the contact with theartificial stomach acid were measured by using Lactobacilli MRS Agar(DIFCO) to calculate viability (%).

The selection criterion, although varying depending on bacterialspecies, was set to 0.5 or more, because all the bacilli selected in thepresent case were Lactobacillus gasseri. All of lactobacilli forblood-group A yogurt (L. gasseri OLL 2804 and L. gasseri OLL 2818), forblood-group O yogurt (L. gasseri OLL 2827 and L. gasseri OLL 2915), andfor blood group B yogurt (L. gasseri OLL 2877 and L. gasseri OLL 2901),fulfilled the above criterion (Table 1).

3-(2) Bile-Acid Tolerance Test

Ten micro litters of lactobacilli prepared by two activation cultures inLactobacilli MRS broth (DIFCO) (37° C., 18 hours) was inoculated to 5 mLof Lactobacilli MRS broth (DIFCO) containing 0.9% Bacto-Oxgall (DIFCO),and then anaerobically cultured at 37° C. At 18 hours after the culture,medium turbidity (OD₆₅₀) was measured.

The selection criterion, although varying depending on bacterialspecies, was set to 0.08 or more with lactobacilli for blood-group Ayogurt and lactobacilli for blood-group O yogurt. Both of lactobacillifor blood-group A yogurt (L. gasseri OLL 2804 and L. gasseri OLL 2818)and lactobacilli for blood-group O yogurt (L. gasseri OLL 2827 and L.gasseri OLL 2915) fulfilled the above criterion (Table 1). Amonglactobacilli for blood-group B yogurt, the bile-acid tolerance of L.gasseri OLL2901 was 0.031, but this is considered to be within a rangethat causes no special problem regarding retention in the intestine.Scientific properties of the above-described six bacillus strains areshown in Table 2.

TABLE 2 Blood Group A Blood Group O Blood Group B 2804 Strain 2818Strain 2827 Strain 2915 Strain 2877 Strain 2901 Strain Features of Roundshape Round shape Round shape Round shape Round shape Round shapecolonies on Light yellow Light yellow Light yellow Light yellow Lightyellow Light yellow medium Smooth Smooth Smooth Smooth Smooth Smooth(Lactobacillus Flat Flat Flat Flat Flat Flat MRS Ager, DIFCO) Bacterialshape Bacilliform Bacilliform Bacilliform Bacilliform BacilliformBacilliform Gram stain Positive Positive Positive Positive PositivePositive Type of lactate Homolactic Homolactic Homolactic HomolacticHomolactic Homolactic fermentation fermentation fermentationfermentation fermentation fermentation fermentation Aerobicgrowth + + + + + + Temperature for 15° C.− 15° C.− 15° C.− 15° C.− 15°C.− 15° C.− growth 45° C.+ 45° C.+ 45° C.+ 45° C.+ 45° C.+ 45° C.+ SugarArabinose − − − − − − fermentability Xylose − − − − − − Rhamnose − − − −− − Ribose − − − − − − Glucose + + + + + + Mannose + + + + + +Fructose + + + + + + Galactose + + + + + + Sucrose + + + + + +Cellobiose + + + + + + Lactose + − + − − + Trehalose + + + + + +Melibiose + − − − − − Raffinose − − − − − − Melezitose − − − − − −Mannitol − − − − − − Sorbitol + − − − − − Gas production − − − − − −Stomach-acid 24.906 6.212 0.959 0.585 10.812 8.923 tolerance (survivalrate, %) Bile-acid 0.119 0.117 0.118 0.086 0.651 0.031 tolerance (OD₆₅₀)

Example 4 Production of Yogurt Using Blood Group-Specifically-BindingLactobacilli

Yogurt was produced by using the lactobacilli (lactobacilli for yogurtfor blood group A: L. gasseri OLL 2804 and L. gasseri OLL 2818,lactobacilli for yogurt for blood group O: L. gasseri OLL 2827 and L.gasseri OLL 2915) selected as mentioned above.

4-(1) An Example of Yogurt Production Using L. gasseri OLL 12804

Plain yogurt was prepared by using L. gasseri OLL 2804 strain. First,bulk starters were prepared by inoculating L. gasseri OLL 2804 strain,L. bulgaricus JCM 1002^(T), and S. thermophilus ATCC 19258 at 1% each to10% nonfat-dry-milk media and culturing them at 37° C. for 15 hours.

The starters of L. bulgaricus JCM 1002^(T) and S. thermophilus ATCC19258 were inoculated at 1% each and the starter of L. gasseri OLL 2804strain was inoculated at 5% to a yogurt mix (SNF: 9.5%, FAT: 3.0%) thathad been heat-treated at 95° C. for five minutes, and fermentation wascarried out at 43° C. for four hours.

Numbers of live L. gasseri OLL 2804, L. bulgaricus JCM 1002^(T), and S.thermophilus ATCC 19258, immediately after fermentation and cooling,were 12.5×10⁷ CFU/mL, 14.0×10⁷ CFU/mL, and 11.8×10⁸ CFU/mL,respectively, and both the flavor and physical properties were good.When the above-mentioned yogurt was preserved at 10° C., numbers of livebacilli on day 25 of preservation were 9.60×10⁷ CFU/mL for L. gasseriOLL 2804, 7.50×10⁷ CFU/mL for L. bulgaricus JCM 1002^(T), and 11.0×10⁷CFU/mL for S. thermophilus ATCC 19258. The decrease in live L. gasseriOLL 2804 number was small as shown by its survival rate being 77% of thelive bacillus number on day 1 of the preservation. Flavor and physicalproperties of the preserved product were also good.

4-(2) An Example of Yogurt Production Using L. gasseri OLL 2818

Plain yogurt was prepared by using L. gasseri OLL 2818 strain. First,bulk starters were prepared by inoculating L. gasseri OLL 2818 strain,L. bulgaricus JCM 1002^(T), and S. thermophilus ATCC 19258 at 1% each to10% nonfat-dry-milk media and culturing them at 37° C. for 15 hours.

The starters of L. bulgaricus JCM 1002^(T) and S. thermophilus ATCC19258 were inoculated at 1% each and the starter of L. gasseri OLL 2818strain was inoculated at 5% to a yogurt mix (SNF: 9.5%. FAT: 3.0%) thathad been heat-treated at 95° C. for five minutes, and fermentation wascarried out at 43° C. for four hours.

Numbers of live L. gasseri OLL 2818, L. bulgaricus JCM 1002^(T), and S.thermophilus ATCC 19258, immediately after fermentation and cooling,were 14.0×10⁷ CFU/mL, 20.0×10⁷ CFU/mL, and 11.4×10⁸ CFU/mL,respectively, and both the flavor and physical properties were good.When this yogurt was preserved at 10° C., numbers of live bacilli on day25 of preservation were 11.4×10⁷ CFU/mL for L. gasseri OLL 2818,7.00×10⁷ CFU/mL for L. bulgaricus JCM 1002^(T), and 10.0×10⁷ CFU/mL forS. thermophilus ATCC 19258. The decrease in live L. gasseri OLL 2818number was small as shown by the survival rate being 82% of the livebacillus number on day 1 of preservation. The flavor and physicalproperties of the preserved product were also good.

INDUSTRIAL APPLICABILITY

The present invention provides human ABO blood group-compatiblelactobacilli and methods for screening them. The lactobacilli of thepresent invention or the lactobacillus strains obtained by the screeningmethods of the present invention are lactobacilli that have a highability to bind to the intestine of each of the ABO-blood groups. Byapplying these lactobacilli to the production of food and drinkproducts, it becomes possible to provide new probiotic food and drinkproducts, including yogurt with blood group-specific functions.

1. A method of screening for a human blood group A-compatiblelactobacillus, comprising: (a) providing a candidate lactobacillus; (b)determining ability of the candidate lactobacillus to recognize a humanblood group A antigen, a human blood group B antigen, and a human bloodgroup H antigen, wherein the recognizing ability is determined bymeasuring response units (RU) of surface plasmon resonance spectrum,wherein the human blood group A antigen comprises [GalNAcα1-3(Fucα1-2)Gal-]; and (c) selecting the candidate lactobacillus as a humanblood group A-compatible lactobacillus if: (i) blood group Aantigen-recognizing ability is 2,000 RU or higher; (ii) blood group Bantigen-recognizing ability is 100 RU or lower; and (iii) blood group Hantigen-recognizing ability is 100 RU or lower.
 2. The method, accordingto claim 1, comprising selecting the candidate lactobacillus as a humanblood group A-compatible lactobacillus if ratio of the blood group Aantigen-recognizing ability to the blood group B antigen-recognizingability is 70 or higher.
 3. The method, according to claim 1, comprisingselecting the candidate lactobacillus as a human blood groupA-compatible lactobacillus if ratio of the blood group Aantigen-recognizing ability to the blood group H antigen-recognizingability is 100 or higher.
 4. The method, according to claim 1,comprising: determining ability of the candidate lactobacillus torecognize A-human colonic mucin (A-HCM) and O-human colonic mucin(O-HCM) using surface plasmon resonance; and selecting the candidatelactobacillus as a human blood group A-compatible lactobacillus ifA-HCM-recognizing ability is higher than O-HCM-recognizing ability. 5.The method, according to claim 4, comprising selecting the candidatelactobacillus as a human blood group A-compatible lactobacillus if: theA-HCM-recognizing ability is 100 RU or higher; and the O-HCM-recognizingability is 100 RU or lower.
 6. The method, according to claim 1, whereinthe human blood group B antigen comprises [Galα1-3(Fucα1-2)Gal-].
 7. Themethod, according to claim 1, wherein the human blood group H antigencomprises [Fucα1-2Gal-].
 8. A method of screening for a human bloodgroup B-compatible lactobacillus, comprising: (a) providing a candidatelactobacillus; (b) determining ability of the candidate lactobacillus torecognize a human blood group A antigen, a human blood group B antigen,and a human blood group H antigen using surface plasmon resonance,wherein the recognizing ability is determined by measuring responseunits (RU) of surface plasmon resonance spectrum, wherein the humanblood group B antigen comprises [Galα1-3 (Fucα1-2)Gal-]; and (c)selecting the candidate lactobacillus as a human blood groupB-compatible lactobacillus if: (i) blood group B antigen-recognizingability is 2,000 RU or higher; (ii) blood group A antigen-recognizingability is 100 RU or lower; and (iii) blood group H antigen-recognizingability is 100 RU or lower.
 9. The method, according to claim 8,comprising selecting the candidate lactobacillus as a human blood groupB-compatible lactobacillus if ratio of the blood group Bantigen-recognizing ability to the blood group A antigen-recognizingability is 50 or higher.
 10. The method, according to claim 8,comprising selecting the candidate lactobacillus as a human blood groupB-compatible lactobacillus if ratio of the blood group Bantigen-recognizing ability to the blood group H antigen-recognizingability is 20 or higher.
 11. The method, according to claim 8,comprising: determining ability of the candidate lactobacillus torecognize A-human colonic mucin (A-HCM), B-human colonic mucin (B-HCM),and O-human colonic mucin (O-HCM) using surface plasmon resonance; andselecting the candidate lactobacillus as a human blood groupB-compatible lactobacillus if B-HCM-recognizing ability is higher thanA-HCM-recognizing ability and O-HCM-recognizing ability.
 12. The method,according to claim 8, wherein the human blood group A antigen comprises[GalNAcα1-3(Fucα1-2)Gal-].
 13. The method, according to claim 8, whereinthe human blood group H antigen comprises [Fucα1-2Gal-].
 14. A method ofscreening for a human blood group O-compatible lactobacillus,comprising: (a) providing a candidate lactobacillus; (b) determiningability of the candidate lactobacillus to recognize a human blood groupA antigen, a human group B antigen, and a human group H antigen usingsurface plasmon resonance, wherein the recognizing ability is determinedby measuring response units (RU) of surface plasmon resonance spectrum,wherein the human blood group H antigen comprises [Fucα1-2Gal-]; and (c)selecting the candidate lactobacillus as a human blood groupO-compatible lactobacillus if: (i) blood group H antigen-recognizingability is 700 RU or higher; (ii) blood group A antigen-recognizingability is 100 RU or lower; and (iii) blood group B antigen-recognizingability is 100 RU or lower.
 15. The method, according to claim 14,comprising selecting the candidate lactobacillus as a human blood groupO-compatible lactobacillus if ratio of the blood group Hantigen-recognizing ability to the blood group A antigen-recognizingability is 800 or higher.
 16. The method, according to claim 14,comprising selecting the candidate lactobacillus as a human blood groupO-compatible lactobacillus if ratio of the blood group Hantigen-recognizing ability to the blood group B antigen-recognizingability is 20 or higher.
 17. The method, according to claim 14,comprising: determining ability of the candidate lactobacillus torecognize A-human colonic mucin (A-HCM) and O-human colonic mucin(O-HCM) using surface plasmon resonance; and selecting the candidatelactobacillus as a human blood group O-compatible lactobacillus ifO-HCM-recognizing ability is higher than A-HCM-recognizing ability. 18.The method, according to claim 17, comprising selecting the candidatelactobacillus as a human blood group O-compatible lactobacillus if:O-HCM-recognizing ability is 100 RU or higher; and A-HCM-recognizingability is 1,000 RU or lower.
 19. The method, according to claim 14,wherein the human blood group A antigen comprises[GalNAcα1-3(Fucα1-2)Gal-].
 20. The method, according to claim 14,wherein the human blood group B antigen comprises [Galα1-3(Fucα1-2)G-].